The present invention is generally directed to novel glycosylated steroid derivatives for facilitating the transport of molecules across biological membranes and the blood-brain barrier. The invention is further directed to a novel glycosylation process for the efficient synthesis of these glycosylated steroid derivatives.
The introduction of molecules of diagnostic, prophylactic, or therapeutic interest (termed herein "therapeutically-significant-molecules" or "therapeutically-significant-compounds") into cells requires traversal of one or more semipermeable biological membranes. The basic structural unit of biological membranes is a phospholipid bilayer, in which are embedded proteins of various size and composition. The surfaces of the phospholipid bilayer, which project into the aqueous cellular environment, are formed by the hydrophilic heads of the phospholipids; the interior, by the fatty acyl hydrophobic tails. The membrane proteins may be involved in transport processes and also may serve as receptors in cellular regulatory mechanisms.
Natural mechanisms for traversal of biological membranes include passive diffusion, facilitated diffusion, active transport, receptor-mediated endocytosis and pinocytosis. Passive diffusion works best for small molecules which are lipid-soluble. However, biological membranes are essentially impermeable to most water-soluble molecules, such as nucleosides, amino acids, proteins, and other hydrophilic, therapeutically-significant molecules. Such molecules enter cells via some type of carrier-mediated transport system in which specific compounds facilitate traversal of the membrane. Natural carriers for facilitating traversal of the membrane are of limited utility, however, as such carriers will accept substrates of only a predetermined molecular configuration.
Specific strategies also have been proposed for introducing molecules, particularly oligonucleotides, into the cell nucleus. Among the techniques reported are utilization of HIV Tat protein-drug conjugates [WO 91/09958] and utilization of oligonucleotide-cholesterol conjugates [Letsinger R.L. et al. "Cholesteryl-conjugated oligonucleotides: Synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture." Proc. Natl. Acad. Sci. USA 86: 6553-6556 (September 1989); Stein C. A. et al. "Mode of Action of 5'-Linked Cholesteryl Phosphorothioate Oligodeoxynucleotides in Inhibiting Syncytia Formation and Infection by HIV-1 and HIV-2 in Vitro." Biochemistry 30: 2439-2444 (1991)].
Targeting molecules to the brain requires traversal of the blood-brain barrier--a capillary-including system, with unique morphological characteristics, which acts as a systemwide cellular membrane separating the brain interstitial space from the blood. Like biological membranes, the blood-brain barrier is relatively impermeable to many hydrophilic, therapeutically-significant-compounds. Among the strategies which have been developed for targeting compounds to the brain are direct delivery by invasive procedures, intra-arterial infusion of hypertonic substances, and conversion of hydrophilic compounds to lipid-soluble entities. Recent attempts at facilitated transport, as described in U.S. Pat. No. 4,902,505, involve coupling a hydrophilic peptide of interest to a peptide carrier which, by itself, is capable of traversing the barrier via receptor-mediated transcytosis.
The carrier compounds of the present invention are novel glycosylated steroid derivatives, soluble in both aqueous and membrane-like environments. Unlike previously-known carriers, the compounds of the present invention may be used to facilitate the transport of a wide variety of molecules, particularly in vivo.
Prior to the present invention, no method existed for synthesizing all of the glycosylated steroid derivatives of the present invention. Many glycosylation reactions using thioglycosides have been reported. [Ferrier R. J. et al. "A Potentially Versatile Synthesis of Glycosides." Carbohydrate Research 27: 55-61 (1973); Garegg P. J. et al. "A reinvestigation of glycosidation reactions using 1-thioglycosides as glycosyl donors and thiophilic cations as promoters." Carbohydrate Research 116: 162-5 (1983); Nicolaou K. C. et al. "A Mild and General Method for the Synthesis of O-Glycosides." J Am Chem Soc 105: 2430-2434 (1983); Lonn H. "Synthesis of a tri- and a hepta-saccharide which contain .alpha.-L-fucopyranosyl groups and are part of the complex type of carbohydrate moiety of glycoproteins." Carbohydrate Research 139: 105-113 (1985); Andersson F. et al. "Synthesis of 1,2-cis-linked glycosides using dimethyl(methylthio)sulfonium triflate as promoter and thioglycosides as glycosyl donors." Tetrahedron Letters pp. 3919-3922 (1986); Brown D. S. et al. "Preparation of cyclic ether acetals from 2-benzenesulphonyl derivatives: a new mild glycosidation procedure." Tetrahedron Letters 29/38: 4873-4876 (1988); Ito Y et al. "Benzeneselenenyl triflate as a promoter of thioglycosides: a new method for O-glycosylation using thioglycosides." Tetrahedron Letters pp. 1061-4 (1988); Dasgupta F. et al. "Alkyl sulfonyl triflate as activator in the thioglycoside-mediated formation of .beta.-glycosidic linkages during oligosaccharide synthesis." Carbohydrate Research 177: c13-c17 (1988)]. However, none of these reported methods teach the use of a glycosyl sulfoxide as a glycosylating agent.
Utilization of an activated glycosyl sulfoxide intermediate in a process for glycosylating steroids, previously has been reported by the inventor in J. Am. Chem. Soc. 111: 6881-2 (1989), the content of which is hereby incorporated by reference. However, the reported method represents only preliminary results on the glycosylation of steroids of the Formula (I). More specifically, further experimentation in the series has revealed unique reaction conditions which are necessary to achieve the efficient and stereo-selective synthesis of glycosylated compounds of the Formula (I). The reaction solvent used plays a critical role in the stereoselectivity of glycosylation. Using a nonpolar, aprotic solvent increases selectivity for alpha (.alpha.) glycosidic bond formation while the use of a polar, aprotic solvent such as propionitrite increases selectivity for beta (.beta.) glycosidic bond formation. The type of sulfoxide used in the glycosylation reaction also affects the stereoselectivity of the reaction. For example, it is vital to use the para-methoxy phenyl sulfoxide as the leaving group in the novel process described herein to obtain beta (.beta.) selectivity in the glycosidic bond formation. The yield of the glycosylation reaction yielding alpha (.alpha.) or beta (.beta.) glycosidic linkages may be increased by the use of less than one equivalent of triflic anhydride in the glycosylation process.
Finally, the protecting groups on the glycosyl donor also have an impact on the stereochemical course of the glycosylation reaction. When the protecting group used on the glycosyl donor is pivaloyl, only beta (.beta.) glycosidic bonds are formed in the glycosylation process, regardless of whether an aprotic, non-polar solvent or an aprotic, polar solvent is used for the reaction. The above factors taken together indicate that one skilled in the art could not have practiced the invention without the detailed further experimentation provided herein.